BOTRYTIS-INDUCED KINASE1, a plasma membrane-localized receptor-like protein kinase, is a negative regulator of phosphate homeostasis in Arabidopsis thaliana
© The Author(s). 2016
Received: 27 January 2016
Accepted: 28 June 2016
Published: 7 July 2016
Plants have evolved complex coordinated regulatory networks to cope with deficiency of phosphate (Pi) in their growth environment; however, the detailed molecular mechanisms that regulate Pi sensing and signaling pathways are not fully understood yet. We report here that the involvement of Arabidopsis BIK1, a plasma membrane-localized receptor-like protein kinase that plays critical role in immunity, in Pi starvation response.
qRT-PCR analysis revealed that expression of BIK1 was induced by Pi starvation and GUS staining indicated that the BIK1 promoter activity was detected in root, stem and leaf tissues of plants grown in Pi starvation condition, demonstrating that BIK1 is responsive to Pi starvation stress. The bik1 plants accumulated higher Pi content in root and leaf tissues and exhibited altered root architecture such as shorter primary roots, longer and more root hairs and lateral roots, as compared with those in the wild type plants, when grown under Pi sufficient and deficient conditions. Increased anthocyanin content and acid phosphatase activity, reduced accumulation of reactive oxygen species and downregulated expression of Pi starvation-induced genes including PHR1, WRKY75, AT4, PHT1;2 and PHT1;4 were observed in bik1 plants grown under Pi deficient condition. Furthermore, the expression of PHO2 was downregulated while the expression of miRNA399a and miRNA399d, which target to PHO2, was upregulated in bik1 plants, compared to the wild type plants, when grown under Pi deficient condition.
Our results demonstrate that BIK1 is a Pi starvation-responsive gene that functions as a negative regulator of Pi homeostasis in Arabidopsis.
KeywordsBOTRYTIS-INDUCED KINASE1 (BIK1) Phosphate starvation stress Phosphate starvation response Root architecture
Phosphate (Pi) is one of the indispensable macronutrients to plants for growth, development and reproduction. Pi deficiency (−Pi) is one of the main limiting factors for increasing crop yield and improving quality because of low bioavailability of Pi in soil [1–4]. To cope with Pi deficiency, plants develop a series of tightly controlled adaptive responses including external developmental alterations of increasing Pi absorption and internal metabolic, physiological and biochemical alterations of reducing Pi usage [4–6]. To increase Pi uptake under Pi depletion stress, plants adapt to modulate the root architecture bearing more and longer lateral roots as well as denser root hairs, which enable the roots to explore the Pi resources in soil [7–12]. To reduce Pi consumption under Pi deficiency condition, plants often modulate metabolisms to maintain intracellular Pi homeostasis by reducing metabolic consumption of Pi [1, 3, 13–15] and degrading compounds to release Pi . Furthermore, a series of physiological and biochemical adaptions including the induction and secretion of phosphatases and organic acids and accumulation of protective metabolites such as anthocyanin help augment the availability of both endogenous and exogenous Pi [1, 4, 17, 18].
Genetic, physiological and biochemical studies in Arabidopsis have demonstrated that the acquisition, allocation, and metabolism of Pi are highly regulated processes and require the concerted action of multiple membrane Pi transport systems [19–23]. Among five distinct classes of proteins possessing Pi transport activity, the plastidic Pi translocator group function as antiport systems, whereas the other four Pi transporter families, named the PHOSPHATE TRANSPORTER1 (Pht1), Pht2, Pht3 and Pht4, contribute to the acquisition, allocation and remobilization of Pi [24–27]. Most of the plasma membrane-localized high affinity transporters in the Pht1 family mediate Pi acquisition from external environment [1, 24, 28–30]. Once Pi is transported into root epidemic cells, translocation and allocation of Pi within the plants and cells are key steps in maintaining Pi homeostasis at cell and whole plant levels. The low affinity transporters in the Pht2 and Pht4 families are thought to participate mainly in Pi transfer across internal cellular membranes and thus allocate Pi in different compartments of the cells [25, 27, 31–33]. Pht1;5, a Pht1 family member, PHO1, Pht1;8 and Pht1;9 were shown to play critical roles in systemic regulation of Pi homeostasis, e.g. mobilization of Pi from source to sink organs in accordance with the Pi status of the plant [34–36].
Functional characterization of genes in a number of mutants with altered response to Pi depletion have led to the identification of several different regulatory mechanisms controlling the adaptive responses to Pi starvation. These regulatory mechanisms include transcriptional regulation by transcription factors such as PHR1, WRKY6, WRKY42, WRKY45, WRKY75, ZAT6, bHLH32, ERF070 and MYB62 [37–47], posttranscriptional regulation by microRNAs including miRNA399 [48–53], posttranslational regulation by protein modifications such as sumoylation of SIZ1 , phosphorylation of Pht1.1 and activation of MKK9-MPK3/MPK6 module [55, 56], deubiquitination of UBP14  and chromatin histone modification and epigenetic [46, 58]. Furthermore, it was also demonstrated that Pi starvation response cross-talks signaling mediated by different hormones such as auxin [10, 15, 59–61], cytokinin [10, 15, 62, 63], ethylene [9, 10, 64–66] and gibberellin . Although great advance on the adaptive response to Pi starvation has been made during the last decade, the molecular mechanism that regulates these adaptive processes has yet to be elucidated in detail.
The Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) encodes a plasma membrane-localized receptor-like protein kinase and plays critical roles in Botrytis cinerea resistance . Recent studies have shown that BIK1 interacts with receptors for pathogen- or damage-associated molecular patterns such as FLS2 and PEPRs to regulate immune response against different types of pathogens [69–73] and defense response to insect pests . The bik1 mutant plants showed an altered root architecture , similar to morphological phenotypes often seen in mutants with Pi starvation response , indicating a possible involvement of BIK1 in Pi starvation response in Arabidopsis. Therefore, we investigated whether BIK1 functions in Pi starvation response and our results demonstrate that BIK1 plays a role in regulation of Pi homeostasis in Arabidopsis.
Responsiveness of BIK1 to Pi starvation
Increased Pi concentration in bik1 plants
Altered root architecture in bik1 plants
Increased anthocyanin accumulation and acid phosphatase activity in bik1 plants
Reduced ROS accumulation in bik1 plants under Pi starvation condition
Altered expression of Pi starvation-induced genes and miRNA399 in bik1 plants
It was found that expression of PHO2, encoding a ubiquitin-conjugating E2 enzyme, was suppressed by miRNA399, which controls Pi homeostasis in plants and whose expression is up-regulated by Pi starvation [48–52]. To examine whether PHO2/miRNA399 links to increased accumulation of Pi in bik1 plants, we analyzed the changes in levels of PHO2 expression and two miRNA399 primary transcripts, miRNA399a and miRNA399d, in bik1 seedlings grown under + Pi and –Pi conditions. The expression level of PHO2 and the transcript levels of miRNA399a and miRNA399d were comparable in WT and bik1 seedlings grown under + Pi condition. However, the expression levels of PHO in WT and bik1 seedlings under –Pi condition were significantly reduced whereas the transcript levels of miRNA399a and miRNA399d in WT and bik1 seedlings grown under –Pi condition were markedly increased, as compared with the corresponding seedlings under + Pi condition (Fig. 6b). A further reduction in the expression of PHO2 and a further increase in the transcripts of miRNA399a and miRNA399d in bik1 seedlings was observed as compared with those in WT seedlings grown under –Pi condition (Fig. 6b). Together, these data indicate that the PHO2/miRNA399 plays a role in regulating Pi accumulation in bik1 plants under –Pi condition.
The maintenance of cellular Pi homeostasis in plants involves complicated regulatory mechanisms. Several studies have demonstrated that posttranslational modifications such as phosphorylation, sumoylation and ubiquitination of regulatory proteins play critical roles in Pi starvation responses [54, 55, 57]. Recently, genome-wide co-expression analysis identifies dozens of protein kinases as important regulators of Pi deficiency-induced root hair remodeling [79, 80]. It was also found that in vivo phosphorylation activation of PPC1 (phosphoenolpyruvate carboxylase 1) is involved in the metabolic adaptations of Pi starvation  and activation of MKK9-MPK3/MPK6 enhances phosphate acquisition in Arabidopsis . These findings indicate an important role for protein kinases in regulating Pi starvation response. In this study, we found that BIK1, a plasma membrane-localized receptor-like protein kinase , plays an important role in modulating Pi starvation responses and functions as a negative regulator of Pi homeostasis in Arabidopsis plants. Thus, evidence presented in the current study renders BIK1 for a novel function in Pi starvation response, in addition to its previously reported functions in immunity [68–73].
Several lines of evidence presented in this study support that BIK1 functions in Pi starvation response. Earlier study has shown that expression of BIK1 could be induced by infection with B. cinerea as well as treatments with some well-known defense signaling molecules . Our qRT-PCR analysis of BIK1 expression and determination of BIK1 promoter activity in stable BIK1 pro ::GUS transgenic seedlings demonstrate that BIK1 can be induced by Pi starvation and Pi starvation-induced expression of BIK1 in root and shoot tissues initiated earlier than in leaf tissues (Fig. 1a). The pattern of BIK1 expression induced by Pi starvation is similar to most of the Pi starvation-induced genes identified so far, like WKRY75, ERF070 and ZAT6 [39, 40, 44]. Importantly, promoter activity analysis revealed that GUS staining driven by the BIK1 promoter was initiated in vascular tissues of root (Fig. 1b) that is the primary organ that responds to Pi starvation, further confirming the responsiveness of BIK1 to Pi starvation. Thus, it is likely that BIK1 can respond rapidly to the altered Pi status of plants under Pi starvation condition. It was particularly noteworthy that BIK1 is a plasma membrane-localized receptor-like protein kinase and has been shown to be phosphorylated by other kinases (e.g. BAK1 and FLS2) for its full activity in plant immune responses [69, 70]. In this context, it is thus possible that BIK1 is phosphorylated by unknown kinase during Pi starvation stress and phosphorylated BIK1 acts in Pi starvation response, in addition to its transcriptional regulation upon Pi starvation.
The primary root, lateral root, and root hairs are three main components of the root architecture that is critical to absorption of Pi from soil. Low P availability can drastically alter the root architecture by switching the indeterminate growth to determinate growth to promote lateral root growth . Generally, not only the primary roots but also almost all mature lateral roots enter into the determinate developmental program under low Pi condition . The bik1 seedlings showed a significant change in root architecture and the primary root length of bik1 plants was significantly decreased (Fig. 6). This is similar to a common phenomenon observed that the primary root length is decreased significantly due to the Pi starvation-induced determinate growth in primary root . In addition to the change of the primary root, the bik1 plants showed increased number and length of lateral roots and root hairs (Fig. 6), which increase root surface contacting an increased soil volume to explore Pi availability in soil . Similar root architectures were observed in the bik1 plants under + Pi and –Pi conditions (Fig. 6). Thus, it is likely that the effect of BIK1 on the development of root architecture is independent of the Pi status in plants and Pi availability in soil. Nevertheless, the characteristic root architecture observed in bik1 plants suggests that BIK1 is a negative regulator of lateral root and root hair development. This is in agreement with previous observations that the bik1 plants showed some defects in growth and development, e.g. weak stem strength, early flowering and less seed setting . Together, these data further demonstrate that BIK1 is required for normal plant growth and development . It will be interesting to investigate whether the growth and developmental defects in bik1 plants is due to an activated Pi starvation response and if this is the case, the results obtained will further support a cross-talk between Pi starvation response and plant growth/development. It was previously found that overexpression of ZAT6 retards growth and results in typical Pi starvation responses .
Accompanied with high contents of total Pi in roots and leaves of bik1 plants under + Pi and –Pi conditions (Fig. 2) are the significant accumulation of anthocyanin (Fig. 4a and b), increased activity of acid phosphatase (Fig. 4a and b) and alterations in expression of Pi starvation-induced genes (Fig. 6). Collectively, these data imply that, whatever grown under + Pi condition or under –Pi condition, a Pi starvation response including those of physiological, molecular and metabolic changes is activated in bik1 plants. Increased activity of acid phosphatases in bik1 plants grown under –Pi condition (Fig. 3b) not only represents a characteristic Pi starvation response  but also can release more Pi available for absorption by the roots. This is supported by the findings that mutations in genes encoding for purple acid phosphatases affect markedly the uptake of Pi from exogenous sources [84–86]. Because expression of BIK1 was rapidly induced by Pi starvation (Fig. 1), it seems possible that BIK1 is involved in Pi uptake by regulating the activity of acid phosphatase. Similar observations were also obtained for some Pi starvation response regulators such as PHR1 and ZAT6, which have been shown to affect activity of acid phosphatase and Pi uptake in transgenic plants with altered expression of these genes [40, 85, 87, 88]. Interestingly, expression of Pht1;2 and Pht1;4, encoding for high-affinity Pi transporters [24, 28], WRKY75, encoding a WRKY transcriptional factor involved in Pi acquisition , At4, a member of the Mt4/TPSI1 gene family involved in Pi distribution , and PHR1, encoding a MYB transcriptional factor involved in Pi starvation response signaling , was suppressed significantly in roots of bik1 plants under –Pi condition (Fig. 6a). These data demonstrate that BIK1 has global effect on a set of Pi starvation-induced genes that are involved in Pi acquisition and mobilization. Notably, the expression levels of the tested Pi starvation-responsive gene except PHR1 in bik1 plants were slightly reduced as compared to those in WT plants under + Pi condition (Fig. 6a), indicating a possibility that these Pi starvation-responsive genes may be also affected by mutation of BIK1 itself. Particularly, the expression of PHR1 in bik1 plants grown under + Pi condition was significantly reduced as compared to that in WT plants (Fig. 6a), leading to an open question whether BIK1 acts upstream of PHR1 in regulating the Pi starvation signaling. This can be clarified by phenotyping and analysis of PHR1 expression in BIK1-overexpressing plants and/or detailed examination of biochemical and genetic requirement of BIK1 for Pi starvation response. It is currently difficult to link the function of BIK1 to Pi uptake or its root-to-leaf translocation as Pi uptake and transportation in WT plants is also affected under –Pi condition (Fig. 2). This can further be explained by the reduced expression of these tested Pi starvation-induced genes in root of bik1 plants as compared to those in WT plants under –Pi condition (Fig. 6). Furthermore, altered expression patterns of PHO2 and miRNA399a/d, which are thought to be involved in systemic signaling of Pi starvation response and Pi distribution in plants [49, 52], were also observed in bik1 plants grown under + Pi and –Pi conditions (Fig. 6b). Thus, it is likely that BIK1 is involved in maintaining Pi homeostasis in whole plants. Taken together, these data support an idea that BIK1 is a negative regulator of Pi starvation responses, probably through affecting development of root architecture and a series of physiological and biochemical events related to Pi acquisition, mobilization and translocation. It is reasonable to speculate that, under Pi starvation stress, the bik1 plants may experience a reduced Pi content as the WT plants but they can uptake efficiently Pi from growth environment with their significantly increased root surface area, leading to an increased Pi content in root and leaf tissues of bik1 plants (Fig. 2). However, the detailed mechanism that BIK1 functions in Pi starvation response remains to be explored further. Particularly, characterization of the targets that are phosphorylated by BIK1 will be greatly helpful in elucidating the early signaling events that determine Pi starvation response.
ROS not only are toxic compounds produced in plant response to various stresses but also plays an integral role as signaling molecules in regulation of numerous biological processes such as growth, development, and responses to biotic and/or abiotic stress [89–91]. The involvement of ROS in Pi starvation response has been established recently. In this study, we found that both WT and bik1 plants grown under –Pi condition accumulated more ROS, represented by H2O2 and superoxide anion, than those in plants grown under + Pi condition (Fig. 5). This is similar to the previous observations that H2O2 concentrations in roots increased upon Pi deprivation . Notably, the accumulation of superoxide anion in WT and bik1 plants under + Pi condition was comparable, the accumulation of superoxide anion in bik1 plants was significantly reduced as compared to that in WT plants under –Pi condition (Fig. 5). By contrast, the bik1 plants accumulated lower level of H2O2 than WT plants grown under + Pi condition (Fig. 5), which is similar to the histochemical staining in soil-grown bik1 plants . Thus, it is likely that BIK1 affects the accumulation of superoxide anion and H2O2 in different ways: loss of BIK1 function suppressed, at least partially, the Pi starvation-induced accumulation of superoxide anion while BIK1 regulates directly the accumulation of H2O2 in plants under normal condition. This may also imply different functions of superoxide anion and H2O2 in Pi starvation response. On the other hand, increased accumulation of ROS in plants under –Pi condition might be one of the stress responses as those in response to other abiotic stress . It was recently shown that localization pattern of ROS accumulated in root during Pi starvation stress is critical to shape the root architecture [93–96]. The reduced accumulation of ROS in bik1 plants in relative to those in WT plants (Fig. 5), which is similar to the observation that loss of BIK1 function impaired the PAMP-induced ROS burst in immunity [97, 98], may attribute to the altered root architecture of bik1 plants (Fig. 3). However, detailed analysis of ROS localization and the possible mechanism regulating ROS generation in root of bik1 plants will provide new insights into the connection between BIK1 and ROS in development of root architecture.
In summary, this study demonstrates that BIK1 is a Pi starvation-responsive gene and functions as a negative regulator of Pi homeostasis in Arabidopsis. This not only renders a novel function for BIK1 but also strengthens our understanding of post-transcriptional regulation during Pi starvation responses in plants. Considering that BIK1 is a plasma membrane-localized receptor-like protein kinase, it thus may play a role in sensing and/or processing of the Pi starvation signal during early stage of Pi starvation stress.
Plant materials and growth condition
Arabidopsis thaliana ecotype Columbia (Col-0) and bik1 (provided by Dr. Tesfaye Mengiste, Purdue University, USA) mutant  were used in this study. Seeds were surface sterilized with 70 % ethanol, washed with sterilized distill water and vernalized at 4 °C for 2 days before germination. Hydroponic, solid medium and liquid medium cultivation were used for different purpose of experiments. For solid medium cultivation, the basic medium used contained 2.06 mM NH4NO3, 1.88 mM KNO3, 0.31 mM MgSO4, 0.1 mM MnSO4, 0.03 mM ZnSO4, 0.1 mM CuSO4, 0.3 mM CaCl2, 5.0 mM KI, 0.1 mM CoCl2, 0.1 mM FeSO4; 0.1 mM EDTA, 0.1 mM H3BO3, 1 mM Na2MoO4.2H2O, 3 g/L sucrose, 10 g/L agar, pH 5.8. When treated for Pi sufficiency (+Pi) and Pi deficiency (−Pi), the medium was supplemented with 1 mM KH2PO4, 10 μM KH2PO4 and 0.99 mM KCl. For hydroponic cultivation, seedlings at 5 ~ 7-leaf stage were transferred to 1/2 Hoagland solution for short adaption and then transferred to hydroponic solution containing 250 μM Pi or no Pi . For liquid medium cultivation, seeds were dispensed in 1/2 MS medium without agar and rinsed with sterilized distilled water at 7 days. Seedlings were then transferred in MS liquid medium with Pi (1 mM) or without Pi and allowed to grow with shaking at 85 rpm. Plants were grown in a growth room under a 16 h light (100 μmol · s−1 · m−2 photons m−2 sec−1 of intensity) and 8 h dark cycle at 22 ± 2 °C with 60 % relative humidity.
Measurement of root system architecture
Seven-day-old seedlings grown on 1/2 MS medium were transferred to Petri dishes and treated for + Pi or –Pi for another 7 days. Length of main root, number and length of the lateral root were measured. Total numbers of root hairs in a 5 mm region from root tip were recorded. Data were recorded from 15 individual plants from each treatment.
Measurement of anthocyanin was performed as described previously . Briefly, seedlings grown under + Pi or –Pi condition in solid medium were put into 20 mL extraction solution (propanol : HCl : H2O = 18:1:81, V/V/V) and boiled for 90 s. The mixtures were kept overnight in dark at room temperature, centrifuged for 40 min at 5000× g, and the absorbance (A) was measured at 535 nm and 650 nm. The A535 values were corrected with the A650 values using formula A535 = A535-2.2 × A650. Anthocyanin contents were calculated according to a standard curve prepared with the same protocol.
Quantification of total Pi content
Total Pi content was quantified according to the U.S. Environmental Protection Agency Method 365.2 with minor modifications . Briefly, samples (~50 mg/sample) were flamed to ash after recording of their dry weights and then 100 μL of concentrated HCl was added. Ten microliters of the mixture were drawn and diluted into 790 μL of water, followed by addition of 200 μL assay solution (4.8 mM NH4MoO4, 2.5 N H2SO4, and 35 μM ascorbic acid). The reactions were incubated at 45 °C for 20 min and the absorbance at 650 nm was measured spectrophotometrically. Contents of total Pi were calculated according to the Pi standard curve prepared with the same procedure.
Quantification and staining of acid phosphatase activity
Activity of acid phosphatase was quantified using the pNPP hydrolysis assay according to a previously described method . Briefly, 30 mg of samples were grounded, transferred to Eppendorf tubes and then spin for 10 min at 2000 × g. Reactions containing 100 μL supernatant, 100 μL p-nitrophenol sodium phosphate and 2.8 mL buffer were kept at 30 °C for 10 min, with shaking occasionally, and terminated by addition of 1 mL 0.5 M NaOH. The absorbance of the reactions was determined spectrophotometrically at 400 nm and the activity of acid phosphatase was calculated from the production of p-nitrophenol. Total protein was estimated using Bradford’s reagent and the total acid phosphatase activity was expressed as mU/mg protein. Staining of acid phosphatase activity was performed as described by Tomscha et al.  with minor modifications. Ten-day-old seedlings grown in liquid medium were rinsed in –Pi medium, transferred to fresh medium supplemented with Pi (1 mM) or without Pi and then covered with a layer of 0.05 % agarose solution containing 0.008 % 5-bromo-4-chloro-3-indolyl phosphate (BCIP) .
Detection and quantification of H2O2
For quantification of H2O2, 30 mg samples from 4-week-old plants were completely ground, followed by addition of 200 μL 20 mM K2HPO4 (pH6.5) phosphate buffer. Quantification of H2O2 was performed using a commericial kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufactruer’s recommendation. Detection of superoxide anion was performed by the nitroblue tetrazolium (NBT) staining . Four-week-old seedlings were vacuum infiltrated with 2 ml of 10 mM potassium phosphate buffer (pH 7.5) containing 10 mM NaN3 and 0.1 % NBT for 30 min, cleared by boiling in 96 % ethanol, remained in 50 % ethanol before taking photos.
qRT-PCR analysis of gene expression
Total RNA was extracted using TRIZOL reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. First strand cDNA was synthesized from 500 ng of total RNA using SuperScript III Kit (Invitrogen, Shanghai, China). The qPCR reactions contained 12.5 μL SYBR Premix Ex TaqTM (TaKaRa, Dalian, China), 0.1 μg cDNA and 7.5 pmol of each of gene-specific primers in 25 μL and were conducted on a CFX96 real-time PCR system (BioRad, Hercules, CA, USA). Gene-specific primers used were as followings: BIK1-q-F, 5′-ACT TAT GGG TAC GCC GCG CCT GAG T-3′; BIK1-q-R, 5′-GGC ACG GAC CAC TTG GTC CA-3′; GUS-q-1F, 5′-AGG TGC ACG GGA ATG TTT CG-3′; GUS-q-1R, 5′-TGT GAG AGT CGC AGA ACA TT-3′; PHR1-q-1F, 5′-GTG ATT GGC ATG AAT GGG CTG AC-3′; PHR1-q-1R, 5′-CGC AAT TCC ACA GAC GGA GAA GG-3′; AT4-q-1F, 5′-GAT CGA AGT TGC CCA AAC GA -3′; AT4-q-1R, 5′-GAG CGA TGA AGA TTG CAT GAA G-3′; WRKY75-q-1F, 5′-GAG AAA TCC ACC GAA AAC TTC GAG CAT AT-3′; WRKY75-q-1R, 5′-GCA TGG TTT TTC TTT TCA ACA CAC GTA AAA TGT A-3′; PHT1.2-q-1F, 5′-AGG GCA AGT CCC TCG AAG AAC T-3′; PHT1.2-q-1R, 5′-ATC AAA CAA ACC ACA AAC AAC TCC ACA T-3′; PHT1.4-q-1F, 5′-TTG CTC CTA ATT TTC CTG ATG CT -3′; PHT1.4-q-1R, 5′-TGT GCC GGC CGA AAT CT-3′. Pri-miRNA399a-1F, 5′-TGG CAG GAA ACC ATT ACT TAG ATC T-3′; Pri-miRNA399a-1R, 5′-TCA CTA ATT AAA AGC AAT GCA TAA AGA GA-3′; Pri-miRNA399d-1F, 5′-TTA CTG GGC GAA TAC TCC TAT GG-3′; Pri-miRNA399d-1R, 5′-ATT TTA CTT GCA TAT CTA GCC AAT GC-3′; PHO2-q-1F, 5′-AGG TTT GAA GCT CCA CCC TCA-3′; PHO2-q-1R, 5′-CCC AAG ATG TGA TTG GAG TTC C-3′; UBQ10-q-1F, 5′-GGC CTT GTA TAA TCC CTG ATG AAT AAG-3′; UBQ10-q-1R, 5′-AAA GAG ATA ACA GGA ACG GAA ACA TAG T-3′. Relative gene expression levels were calculated using 2–△△CT method with three independent biological replicates.
Generation of BIK1 pro ::GUS transgenic line and GUS staining
A 2 kb sequence upstream of the BIK1 start codon was PCR amplified using primers AtBIK1-GUS-1F (5′-ATA CTG CAG CTT GTT GAT TGA TTA ATA GAT TAC C-3, a PstI site in bold) and AtBIK1-GUS-1R (5′-GCC GGA TCC AGA ACT GAA GCA AGA ACC CAT C-3′, a BamHI site in bold) and cloned into vector pCAMBIA1301. Transformation of wild-type Col-0 plants was performed using the floral dip infiltration method mediated by Agrobacterium tumefaciens strain GV3101. Plants of T2 generations from kanamycin-resistant transformants were used for GUS histochemical staining .
+Pi, Pi sufficiency; B. cinerea, Botrytis cinerea; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; BIK1, Botrytis-induced kinase1; MKK, mitogen-activated protein kinase kinase; MPK, mitogen-activated protein kinase; NBT, nitroblue tetrazolium; PAMP, pathogen associated molecular pattern; Pht1, PHOSPHATE TRANSPORTER1; Pi, phosphate; −Pi, Pi deficiency; pNPP, p-nitrophenol sodium phosphate; PPC1, phosphoenolpyruvate carboxylase 1; qRT-PCR, quantitative reverse transcription PCR; ROS, reactive oxygen species; WT, wild type
We are grateful to Dr. Tesfaye Mengiste, Purdue University, USA, for providing the Arabidopsis WT and bik1 seeds used in this study.
This work was supported by the National Natural Foundation of Sciences (no. 31272028) and the Ph.D. Program Foundation of Ministry of Education of China (no. 20120101110070).
Availability of supporting data
All data supporting the results of this article are included within the article.
HZ and FS designed the study and all experiments; HZ, LH and YH conducted all the molecular biology and physiology experiments; HZ and FS analyzed the data; FS drafted the manuscript with HZ. All authors have read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Not applicable. No human or animals were involved in this study.
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